Artemisinin-based combination therapies have been pivotal in achieving major reductions in the global burden of Plasmodium falciparum (Pf) malaria. Pf parasite resistance to artemisinin, which first emerged in Cambodia through mutations in K13, has placed increased selection pressure on its partner drugs. Recent reports document clinical treatment failures with the first-line therapy dihydroartemisinin+piperaquine (PPQ) in Cambodia. PPQ resistance is also emerging in French Guiana. The absence of fully effective alternatives underscores the need to define the molecular basis of PPQ resistance as a means to curtail its spread. Whole-genome sequence (WGS) analysis and phenotypic studies of PPQ-resistant Cambodian isolates have identified candidate genes whose putative functions relate to hemoglobin (Hb) metabolism and solute transport in the parasite's digestive vacuole (DV). Our central hypothesis is that PPQ resistance is mediated by altered Hb metabolism in the DV and accompanying changes in transporters that regulate DV physiology. In Aim 1 we address the hypothesis that Cambodian isolates achieve resistance primarily via amplification of plasmepsins 2 and 3, which encode Hb-digesting proteases. We will also genetically assess the evidence implicating mutations in the Hb peptidase DPAP1 and the DV solute and multidrug transporters PfCRT and PfMDR1. To test this hypothesis, parasites will be engineered through transgene expression and Cas9 or zinc-finger nuclease-mediated gene editing. Changes in resistance will be examined using PPQ survival and dose-response assays. In Aim 2 we tackle the emerging problem of PPQ resistance in French Guiana by implementing genetic crosses with humanized FRG-NOD mice that are receptive to Pf liver and blood stages. WGS and phenotypic analysis of the progeny will identify loci linked to resistance. Parallel WGS studies on PPQ-resistant isolates will help converge on the selection of resistance candidates, whose role will be assessed using transfection. In Aim 3 we define the functional basis of PPQ resistance and its impact on other antimalarials. Using clinical isolates and recombinant lines, we will assess whether reduced PPQ accumulation and heme binding is a hallmark of resistance. We will also evaluate whether resistance results in increased levels of toxic free heme resulting from Hb metabolism. Metabolomic studies will delineate the relationship between PPQ resistance and Hb digestion. Finally, we will assess the impact of resistance on other heme-binding antimalarials, with an emphasis on identifying drugs that retain or even increase their activity against PPQ-resistant parasites. We believe that this project provides powerful and scientifically innovative approaches to elucidate the molecular basis of PPQ resistance, yield genetic markers to monitor its emergence and spread, and identify optimal means of treatment. This project will also provide important new insights into Hb metabolism, which continues to provide a rich source of antimalarials for future clinical use.